Lundin Arne, Eriksson Jonas
BioThema AB, Stationsvagen 17, Handen, Sweden.
Assay Drug Dev Technol. 2008 Aug;6(4):531-41. doi: 10.1089/adt.2008.133.
The firefly luciferin-luciferase reaction has been used to set up an assay for protein kinase based on measuring ATP consumption rate as the first-order rate constant for the kinase reaction. The assay obviates the problems encountered with previous bioluminescent protein kinase assays such as interference with the luciferase reaction from library compounds, nonlinear standard curves, and limited dynamic ranges. In the assay described in the present paper luciferase and luciferin are present during the entire kinase reaction, and the light emission can be measured continuously. In an HTS situation the light emission is measured only twice, i.e., initially and after a predetermined time. After a fivefold reduction of the ATP concentration a Z' value of 0.96 was obtained. Light emission data from samples with kinase are normalized with light emission data from blanks without kinase. First-order rate constants for the kinase reaction calculated from normalized light emission are not affected by a moderate degree of inactivation of luciferase and luciferin during the measuring time. The constants have the same value at all ATP concentrations much lower than the K(m) of the luciferase and the kinase. These factors make the assay very robust and influenced neither by ATP concentration nor by luciferase inhibition. The measuring time depends on the kinase activity and can be varied from minutes to more than 8 h provided the kinase is stable and the evaporation of water from the wells is acceptable. The assay is linear with respect to kinase activity over three orders of magnitude. The new reagents also allowed us to determine K(m) values for ATP and for Kemptide.
萤火虫荧光素 - 荧光素酶反应已被用于建立一种基于测量ATP消耗速率作为激酶反应一级速率常数的蛋白激酶检测方法。该检测方法避免了先前生物发光蛋白激酶检测中遇到的问题，如文库化合物对荧光素酶反应的干扰、非线性标准曲线和有限的动态范围。在本文所述的检测方法中，荧光素酶和荧光素在整个激酶反应过程中都存在，并且可以连续测量发光。在高通量筛选的情况下，仅在初始和预定时间后测量两次发光。ATP浓度降低五倍后，获得的Z'值为0.96。含有激酶的样品的发光数据用不含激酶的空白样品的发光数据进行归一化。根据归一化发光计算的激酶反应一级速率常数在测量时间内不受荧光素酶和荧光素适度失活的影响。在所有远低于荧光素酶和激酶K(m)的ATP浓度下，这些常数具有相同的值。这些因素使得该检测方法非常稳健，不受ATP浓度或荧光素酶抑制的影响。测量时间取决于激酶活性，只要激酶稳定且孔中水分蒸发可接受，测量时间可以从几分钟变化到超过8小时。该检测方法在激酶活性的三个数量级上呈线性关系。新试剂还使我们能够确定ATP和肯普肽的K(m)值。
